Prathusha Kakarla

Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

3D-QSAR AND CONTOUR MAP ANALYSIS OF TARIQUIDAR ANALOGUES AS MULTIDRUG RESISTANCE PROTEIN-1 (MRP1) INHIBITORS

One of the major obstacles to the successful chemotherapy towards several cancers is multidrug resistance of human cancer cells to anti-cancer drugs. An important contributor to multidrug resistance is the human multidrug resistance protein-1 transporter (MRP1), which is an efflux pump of the ABC (ATP binding cassette) superfamily. Thus, highly efficacious, third generation MRP1 inhibitors, like tariquidar analogues, are promising inhibitors of multidrug resistance and are under clinical trials. To maximize the efficacy of MRP1 inhibitors and to reduce systemic toxicity, it is important to limit the exposure of MRP1 inhibitors and anticancer drugs to normal tissues and to increase their co-localization with tumor cells. Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) associated with 3D-Quantitiative structure-activity relationship (3D-QSAR) studies were performed on a series of tariquidar analogues, as selective MDR modulators. Best predictability was obtained with CoMFA model r2 (non-cross-validated square of correlation coefficient) = 0.968, F value = 151.768 with five components, standard error of estimate = 0.107 while the CoMSIA yielded r2 = 0.982, F value = 60.628 with six components, and standard error of estimate = 0.154. These results indicate that steric, electrostatic, hydrophobic (lipophilic), and hydrogen bond donor substituents play significant roles in multidrug resistance modulation of tariquidar analogues upon MRP1. The tariquidar analogue and MRP1 binding and stability data generated from CoMFA and CoMSIA based 3D-contour maps may further aid in study and design of tariquidar analogues as novel, potent and selective MDR modulator drug candidates.

Functional Roles of Highly Conserved Amino Acid Sequence Motifs A and C in Solute Transporters of the Major Facilitator Superfamily

The biological membrane covers all living cells and provides an effective barrier against the passage of biologically important water-soluble solutes. This natural passage barrier is essentially overcome with the use of integral membrane proteins known as solute transporters. These transport systems translocate solutes across the membrane such as in the case of bacterial drug and multidrug resistance efflux pumps. One of the largest groups of transporters is referred to as the major facilitator superfamily. This group contains secondary active transporters such as symporters and antiporters and passive transporters such as uniporters. The transporters within the major facilitator superfamily share conserved structures and primary amino acid sequences. In particular, several highly conserved amino acid sequence motifs have been discovered and studied extensively, providing substantial evidence for their critical functional roles in the transport of solutes across the membrane.

Modulation of the multidrug efflux pump EmrD-3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract

The causative agent of cholera, Vibrio cholerae, is a public health concern. Multidrug-resistant V. cholerae variants may reduce chemotherapeutic efficacies of severe cholera. We previously reported that the multidrug efflux pump EmrD-3 from V. cholerae confers resistance to multiple structurally distinct antimicrobials. Medicinal plant compounds are potential candidates for EmrD-3 efflux pump modulation. The antibacterial activities of garlic Allium sativum, although poorly understood, predicts that a main bioactive component, allyl sulfide, modulates EmrD-3 efflux. Thus, we tested whether A. sativum extract acts in synergy with antimicrobials and that a main bioactive component allyl sulfide inhibits EmrD-3 efflux. We found that A. sativum extract and allyl sulfide inhibited ethidium bromide efflux in cells harboring EmrD-3 and that A. sativum lowered the MICs of multiple antibacterials. We conclude that A. sativum and allyl sulfide inhibit EmrD-3 and that A. sativum extract synergistically enhances antibacterial agents.

Veterinary antibiotics influence trigonelline biosynthesis and plant growth in Arachis hypogaea L.

ABSTRACT Antibiotics from various sources such as livestock waste are being accumulated in the soil. The excessive uptake of antimicrobial agents by plants has been a major concern as it is currently unknown how plants respond to the presence of antibiotics in agricultural lands. The objectives were to analyze the alteration of trigonelline (TRG) biosynthesized by plants in response to various antibiotic stresses and to evaluate the ability of peanut (Arachis hypogaea L.) plants to resist the deleterious impacts of antibiotic uptake. Three veterinary antibiotics used in this study were tetracycline, streptomycin sulfate, and chloramphenicol in the concentrations of 2.5 and 5 mg L−1. Mean TRG amounts were 53.4 ± 1.6 and 59.9 ± 1.1 μg·g−1 dry weight (DW) in Spanish as treated with growth chloramphenicol and streptomycin at 2.5 mg·L−1, respectively, and were significantly (p ≤ .05) different compared to the control (40.4 ± 1.6 μg·g−1 DW) of Spanish. Spanish genotype treated with chloramphenicol at 5 mg·L−1 had a mean TRG amount of 41.0 ± 1.0 μg·g−1 DW and improved yield, with the average pod number of 29.6 ± 7.6 and pod weight of 20.1 ± 6.1 g. TRG was continuously biosynthesized and increased under antibiotic stress up to 12.7% at full pod (R4 growth stage) and 139.1% at beginning maturity (R7), but declined 20.2% at the harvest stage (R8) in all combined genotypes when compared with TRG amounts (21.7 ± 0.6 μg·g−1 DW) at the flowering R1 stage.